脱落酸调控小橡胶粒子蛋白基因表达的分子机制

Natural rubber (NR) is a renewable important strategic material and industrial raw materials. More than 98% of NR in the world come from Hevea brasiliensis. By now, the production of NR is less than one fifth of consumption and the cultivated area is limited in our country. Therefore, improving the rubber yield became the main way for China's current NR production. The traditional method of rubber tree varieties breeding cycle is long, and the effect is slow. Ethylene, as a stimulant of latex production in Hevea brasiliensis, has been widely used in commercial latex production. Intense ethylene stimulation would eventually lead to tapping panel dryness (TPD). Once the TPD occurs, the tapping incision is partly or entirely blocked and the amount of latex production is significantly decreased or stops completely，leading to severe loss in natural rubber production around the world. So, an alternative stimulant instesd of ethylene is in great needs in natural rubber production. Although significant progress has been made on natural rubber biosynthesis and regulation,the molecular mechanism underlying the regulation of natural rubber biosynthesis remain largely unknown. In H. brasilliensis, small rubber particle protein (SRPP) is a key gene involved in rubber biosynthesis. Recent studies have shown that bark treatments with abscisic acid (ABA) cold induce increase latex yield in mature untapped rubber trees.Our previous study found that ABA can significantly improve the small rubber particle protein gene promoter transcription activity。We suspect that ABA increase rubber biosynthesis may through promoted the improvement of the SRPP transcription level, but the specific mechanism is not clear yet.Through this study will focus on the molecular mechanisms of SRPP responsed to ABA. hese researches should enable us to understand further the expressional regulation mechanism of SRPP in natural rubber biosynthesis in Hevea brasiliensis, which may be useful in developing new stimulus to stimulate latex yield in the production of natural rubber.

乙烯利刺激割胶技术对橡胶增产效果显著，但长期使用会造成橡胶树发生死皮、过早衰老和干胶含量下降等弊病，给天然橡胶的生产带来严重负面影响，寻找新型的高产、高效、安全刺激剂已经成为天然橡胶生产中面临的重要课题。已有研究表明脱落酸（ABA）处理成龄未开割橡胶树树干能增加胶乳产量。小橡胶粒子蛋白（SRPP）是橡胶生物合成的关键因子，具有促进胶乳产量增加、提高橡胶合成速率的作用。我们前期研究发现ABA可以显著提高SRPP基因启动子转录活性，推测ABA可能通过促进SRPP转录水平的提高，进而增加橡胶的生物合成，但具体调控机制尚不清楚。本研究将运用生化与分子生物学技术，鉴定SRPP启动子中ABA应答的顺式作用元件和与其互作的转录因子，研究转录因子与SRPP之间的调控关系，阐明ABA调控SRPP的分子机制，为更深入了解橡胶生物合成的调控机制打下基础，为将来应用生物技术提高天然橡胶的产量提供理论依据。

乙烯利刺激割胶技术对橡胶增产效果显著，但长期使用会造成橡胶树发生死皮、过早衰老和干胶含量下降等弊病，给天然橡胶的生产带来严重负面影响，寻找新型的高产、高效、安全刺激剂已经成为天然橡胶生产中面临的重要课题。已有研究表明脱落酸（ABA）处理成龄未开割橡胶树树干能增加胶乳产量。小橡胶粒子蛋白（SRPP）是橡胶生物合成的关键因子，具有促进胶乳产量增加、提高橡胶合成速率的作用。我们前期研究发现ABA 处理可以显著提高SRPP 基因启动子转录活性，推测ABA 可能通过促进SRPP 基因转录水平的提高，进而增加橡胶的生物合成，但具体调控机制尚不清楚。本研究通过对橡胶树SRPP启动子5’端缺失片段进行ABA处理并结合生物信息学分析，确定ABA应答响应元件为MYB1AT和MYCCONSENSUSAT。并将ABA应答响应元件串联重复进行酵母单杂交筛选橡胶树胶乳cDNA文库，得到4个候选转录因子（MYB、 MYC类转录因子各2个）。组织特异表达显示4个转录因子在胶乳中都有较高的表达量，ABA处理可以诱导HbMYB44和HbMYC2b基因的表达。凝胶阻滞实验（EMSA）证实HbMYB44和HbMYC2b蛋白可以与HbSRPP启动子体外结合。双荧光素酶检测结果显示转录因子HblMYB44 和HbMYC2b显著提高了HbSRPP启动子的转录活性。此外，本研究还分离鉴定了橡胶树ABA信号途径相关基因HbPYLs、A类PP2Cs和HbSnRK2s，并进行了组织表达模式分析和激素处理分析。酵母双杂交结果显示，HbMYB44和HbMYC2b可以与HbSnRK2s相互作用。以上研究结果表明，转录因子HbMYB44和HbMYC2b介导了ABA信号途径对HbSRPP基因的调控。该研究为更深入了解橡胶生物合成的调控机制打下基础，为将来应用生物技术提高天然橡胶的产量提供理论依据。